Elbehery Ali H A, Leak David J, Siam Rania
Graduate Program of Biotechnology, The American University in Cairo, Cairo, Egypt.
Department of Biology and Biochemistry, University of Bath, Bath, UK.
Microb Biotechnol. 2017 Jan;10(1):189-202. doi: 10.1111/1751-7915.12468. Epub 2016 Dec 22.
The advent of metagenomics has greatly facilitated the discovery of enzymes with useful biochemical characteristics for industrial and biomedical applications, from environmental niches. In this study, we used sequence-based metagenomics to identify two antibiotic resistance enzymes from the secluded, lower convective layer of Atlantis II Deep Red Sea brine pool (68°C, ~2200 m depth and 250‰ salinity). We assembled > 4 000 000 metagenomic reads, producing 43 555 contigs. Open reading frames (ORFs) called from these contigs were aligned to polypeptides from the Comprehensive Antibiotic Resistance Database using BLASTX. Two ORFs were selected for further analysis. The ORFs putatively coded for 3'-aminoglycoside phosphotransferase [APH(3')] and a class A beta-lactamase (ABL). Both genes were cloned, expressed and characterized for activity and thermal stability. Both enzymes were active in vitro, while only APH(3') was active in vivo. Interestingly, APH(3') proved to be thermostable (T = 61.7°C and ~40% residual activity after 30 min of incubation at 65°C). On the other hand, ABL was not as thermostable, with a T = 43.3°C. In conclusion, we have discovered two novel AR enzymes with potential application as thermophilic selection markers.
宏基因组学的出现极大地促进了从环境生态位中发现具有适用于工业和生物医学应用的有用生化特性的酶。在本研究中，我们使用基于序列的宏基因组学从红海亚特兰蒂斯二号深盐水池（68°C，深度约2200米，盐度250‰）的隐蔽下对流层中鉴定出两种抗生素抗性酶。我们组装了超过400万个宏基因组读数，产生了43555个重叠群。使用BLASTX将从这些重叠群中调用的开放阅读框（ORF）与综合抗生素抗性数据库中的多肽进行比对。选择了两个ORF进行进一步分析。这些ORF推测编码3'-氨基糖苷磷酸转移酶[APH(3')]和A类β-内酰胺酶（ABL）。对这两个基因进行了克隆、表达，并对其活性和热稳定性进行了表征。两种酶在体外均有活性，而只有APH(3')在体内有活性。有趣的是，APH(3')被证明具有热稳定性（T = 61.7°C，在65°C孵育30分钟后残留活性约为40%）。另一方面，ABL的热稳定性较差，T = 43.3°C。总之，我们发现了两种新型抗生素抗性酶，它们有可能作为嗜热选择标记物应用。
